Division of Analytical Biosciences, LACDR, Leiden University, Leiden, The Netherlands.
Mimetas BV, Leiden, The Netherlands.
Sci Rep. 2017 Dec 22;7(1):18071. doi: 10.1038/s41598-017-14716-y.
Current in vitro models to test the barrier function of vasculature are based on flat, two-dimensional monolayers. These monolayers do not have the tubular morphology of vasculature found in vivo and lack important environmental cues from the cellular microenvironment, such as interaction with an extracellular matrix (ECM) and exposure to flow. To increase the physiological relevance of in vitro models of the vasculature, it is crucial to implement these cues and better mimic the native three-dimensional vascular architecture. We established a robust, high-throughput method to culture endothelial cells as 96 three-dimensional and perfusable microvessels and developed a quantitative, real-time permeability assay to assess their barrier function. Culture conditions were optimized for microvessel formation in 7 days and were viable for over 60 days. The microvessels exhibited a permeability to 20 kDa dextran but not to 150 kDa dextran, which mimics the functionality of vasculature in vivo. Also, a dose-dependent effect of VEGF, TNFα and several cytokines confirmed a physiologically relevant response. The throughput and robustness of this method and assay will allow end-users in vascular biology to make the transition from two-dimensional to three-dimensional culture methods to study vasculature.
目前用于测试血管屏障功能的体外模型基于平面二维单层。这些单层没有体内血管的管状形态，也缺乏来自细胞微环境的重要环境线索，例如与细胞外基质（ECM）的相互作用和流动暴露。为了提高血管体外模型的生理相关性，必须实施这些线索，并更好地模拟天然的三维血管结构。我们建立了一种稳健、高通量的方法，可将内皮细胞培养成 96 个三维可灌注的微血管，并开发了一种定量、实时的渗透性测定法来评估其屏障功能。优化了微管形成的培养条件，使其在 7 天内形成，并能维持 60 天以上。微血管对 20 kDa 葡聚糖具有渗透性，但对 150 kDa 葡聚糖没有渗透性，这模拟了体内血管的功能。此外，VEGF、TNFα 和几种细胞因子的剂量依赖性效应证实了具有生理相关性的反应。这种方法和测定法的通量和稳健性将允许血管生物学的终端用户从二维培养方法过渡到三维培养方法来研究血管。
